An understanding of how proteins fold is of major biological importance. This proposal is addressed to the factors which control the folding pathway and thereby define the native structure. The long-term goals of this project are to determine how the amino acid sequence guides the folding process. Knowledge of the "code" which determines the way in which proteins fold would be an invaluable tool for a number of disciplines. In light of the recent advances in genetic engineering, one could, in principle, design a protein with a particular structure and function or redesign a protein in order to make it more stable than its horizon s for the use and production of proteins for medical research and pharmaceuticals. The equilibrium unfolding/refolding and the kinetics of refolding of bovine ribonuclease A derivatives will be studied using absorbance, fluorescence, and high-field 1H nuclear magnetic resonance (NMR). A detailed analysis of intermediate structures along the folding path will be made. Both thermal and chaotropic agent induced structures will be examined. Initial experiments will involve the preparation and characterization of chemically modified derivatives of bovine pancreatic ribonuclease A. In particular, nitrotyrosyl and aminotryrosyl derivatives will be examined. In later experiments the modified residues will serve as structural probes for various portions of the protein molecule and facilitate the assignments of resonances in the 1H NMR spectrum. The unfolding and refolding transitions will be followed and the changes in the signals derived from the probes, along with the known amino acid sequence and native crystal structure, employed to determine changes in structure. Several different sets of unfolding conditions will be examined to determine their effect of the unfolded state structure and the pathway of folding. The project will utilize aqueous-methanol solvent systems to facilitate stabilization of partially-folded protein conformations. This approach is unique and has been quite successful in previous studies.